烟草致癌物NNK活化胰腺癌干细胞Sonic Hedgehog通路诱导慢性胰腺炎癌变的机制研究

Chronic pancreatitis is a well-documented risk factor for pancreatic cancer(PDA）；pancreatic cancer stem cells (PCSC) are the main mediator during chronic pancreatitis canceration. Little is known about the mechanism by which cigarette smoke increase the risk of pancreatic cancer in individuals with chronic pancreatitis. Long-term smoking led to increase the content ofthe NNK in pancreatic tissue microenvironment,which could active the β-adrenergic receptor pathway in pancreatic cancer cell,and promote the pancreatic cancer proliferation.So we hypothesize that tobacco-derived chemicals NNK could active the β adrenergic receptor of PCSC and stimulate G-protein couple kinase-2 phosphorylation, and then transactive the Sonic Hedgehog(SHH) pathway in PCSC, which could led to tumor suppressor gene mutation and the launch of the morphological pancreatic intraepithelial neoplasm (PanIN), it was long suspected that PanIN could progress to invasive pancreatic cancer. In this study, we would reveal the mechanism of PCSC activity and pancreatic carcinogenesis induction by NNK；so we use the laser microdissection, three-dimensional co-culture model and chronic pancreatitis-PanIN-pancreatic cancer model to ducument that NNK could active the PCSC via SHH pathway, then induce INK4A and p53 tumor suppressor gene inactivate, led to pancreatic carcinogenesis ultimately. The study could provide the data to reveal the mechanism of pancreatic carcinogenesis by smoking in the chronic pancreatitis patients.

慢性胰腺炎是胰腺癌的危险因素，胰腺癌干细胞（PCSC）在慢性胰腺炎-胰腺导管上皮瘤变（PanIN）-胰腺癌的转变中发挥核心作用，吸烟可以使慢性胰腺炎癌变风险显著增加。长期吸烟使组织微环境中的致癌物NNK含量增加，可高效激活胰腺癌细胞β肾上腺素能受体（β受体）及其信号通路，促进胰腺癌的发展。本课题假设NNK可激活PCSC的β受体-G蛋白偶联激酶2通路，通过转激活的方式使Sonic Hedgehog（SHH）通路持续活化，导致所分化的胰腺癌细胞抑癌基因失活率增加，最终经PanIN发展为胰腺癌。本课题以吸烟为出发点，以慢性胰腺炎癌变为着眼点，运用体外提取PCSC技术、激光捕获显微切割技术、三维立体共培养模型的构建及大鼠慢性胰腺炎-PanIN-胰腺癌转化模型的构建，证实NNK通过持续活化PCSC的SHH通路诱导INK4A和p53抑癌基因失活并促使胰腺癌发生，探讨吸烟诱导慢性胰腺炎癌变的机制。

吸烟是胰腺癌及慢性胰腺炎的危险因素，可能促进了慢性胰腺炎-胰腺导管上皮瘤变（PanIN）-胰腺癌的转变；在长期吸烟患者，胰腺微环境中的烟草致癌物NNK水平显著升高。NNK是尼古丁的衍生物，是一种高效能的β肾上腺素能受体激动剂；近年研究表明，在肿瘤细胞中β受体激动剂可转激活GRK-2，进一步激活Smo及其转录因子Gli-1。本研究拟通过NNK诱导慢性胰腺炎发生癌前病变，揭示其通过Sonic Hedgehog通路探究其在胰腺癌发生及发展中的作用。.结果：①吸烟能够促进K-ras基因第13密码子突变以及p16基因启动子区甲基化（P<0.05）；在慢性胰腺炎病例中存在p16基因启动子区甲基化及p16低表达；②NNK可以促进慢性胰腺炎大鼠胰腺纤维化及腺泡导管化生；③在NNK促进慢性胰腺炎恶变的过程中，原癌基因K-ras及抑癌基因P16出现异常表达，Gli-1表达增加；④NNK增加胰腺癌细胞的侵袭能力，而propranolol或环巴胺均能抑制NNK的作用；⑤NNK能增加细胞蛋白GRK-2、Smo、Gli-1的表达，联合加入propranolol后能减弱其对三种蛋白的增加作用，NNK联合环巴胺干预细胞则仅能增加蛋白GRK-2的表达。.结论：①吸烟可增加K-ras基因第二外显子第13密码子突变、并且促进p16基因启动子区甲基化，最终导致细胞的恶性表型转换及胰腺癌的发生。②烟草致癌物NNK能成功诱导慢性胰腺炎发生癌前病变，其主要作用机制为NNK通过激活β受体活化GRK-2，进而转激活Sonic Hedgehog信号通路，促进胰腺癌发生；同时可促进HIF-1a生成，可诱导胰腺癌增殖及血管生成，促进胰腺癌进展。